Characterisation of a cluster of genes encoding Theileria annulata AT hook DNA-binding proteins and evidence for localisation to the host cell nucleus

2001 ◽  
Vol 114 (15) ◽  
pp. 2747-2754
Author(s):  
David G. Swan ◽  
Rowena Stern ◽  
Sue McKellar ◽  
Kirsten Phillips ◽  
Chris A. L. Oura ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Cell ◽  
Cell Lines ◽  
Theileria Annulata ◽  
Open Reading Frames ◽  
Nuclear Extracts ◽  
Genes Encoding ◽  
Cell Gene Expression ◽  
Infected Cells ◽  
Host Nucleus

Infection of bovine leukocytes by the apicomplexan parasite Theileria annulata results in alteration of host cell gene expression and stimulation of host cell proliferation. At present, the parasite-derived factors involved in these processes are unknown. Recently, we described the characterisation of a parasite gene (TashAT2), whose polypeptide product bears AT hook DNA-binding motifs and may be transported from the parasite to the host nucleus. We now describe the isolation of a further two genes (TashAT1 and TashAT3) that are very closely related to TashAT2. All three TashAT genes are located together in a tight cluster, interspersed by two further small open reading frames, all facing head to tail. TashAT2 was shown to be expressed in all T. annulata cell lines examined, whereas TashAT1 and TashAT3 were expressed in the sporozoite stage of the parasite, and also in infected cell lines, where their expression was found to vary between different cell lines. Evidence for transport was provided by antisera raised against TashAT1 and TashAT3 that reacted with the host nucleus of T. annulata-infected cells. Reactivity was particularly strong against the host nuclei of the T. annulata-infected cloned cell line D7B12, which is attenuated for differentiation. A polypeptide in the size range predicted for TashAT3 was preferentially detected in host enriched D7B12 nuclear extracts. DNA-binding analysis demonstrated that fusion proteins containing the AT hook region of either TashAT1 or TashAT2 bound preferentially to AT rich DNA.

scholarly journals Host Cell Gene Expression during Human Immunodeficiency Virus Type 1 Latency and Reactivation and Effects of Targeting Genes That Are Differentially Expressed in Viral Latency

2004 ◽  
Vol 78 (17) ◽  
pp. 9458-9473 ◽  
Author(s):  
Vyjayanthi Krishnan ◽  
Steven L. Zeichner
Keyword(s):  
Gene Expression ◽  
Host Cell ◽  
Cell Lines ◽  
Lytic Replication ◽  
Replication Cycle ◽  
Differentially Expressed ◽  
Cellular Genes ◽  
Latently Infected ◽  
Cell Gene Expression ◽  
Infected Cells

ABSTRACT The existence of reservoirs of cells latently infected with human immunodeficiency virus (HIV) is a major obstacle to the elimination of HIV infection. We studied the changes in cellular gene expression that accompany the reactivation and completion of the lytic viral cycle in cell lines chronically infected with HIV-1. We found that several genes exhibited altered expression in the chronically infected cells compared to the uninfected parental cells prior to induction into lytic replication. A number of gene classes showed increased expression in the chronically infected cells, notably including genes encoding proteasomes, histone deacetylases, and many transcription factors. Following induction of the lytic replication cycle, we observed ordered, time-dependent changes in the cellular gene expression pattern. Approximately 1,740 genes, many of which fall into 385 known pathways, were differentially expressed (P < 0.001), indicating that completion of the HIV replication cycle is associated with distinct, temporally ordered changes in host cell gene expression. Maximum changes were observed in the early and intermediate phases of the lytic replication cycle. Since the changes in gene expression in chronically infected cells suggested that cells latently infected with HIV have a different gene expression profile than corresponding uninfected cells, we studied the expression profiles of three different chronically infected cell lines to determine whether they showed similar changes in common cellular genes and pathways. Thirty-two genes showed significant differential expression in all cell lines studied compared to their uninfected parental cell lines. Notable among them were cdc42 and lyn, which were downregulated and are required for HIV Nef binding and viral replication. Other genes previously unrelated to HIV latency or pathogenesis were also differentially expressed. To determine the effects of targeting products of the genes that were differentially expressed in latently infected cells, we treated the latently infected cells with a proteasome inhibitor, clastolactacystin-beta-lactone (CLBL), and an Egr1 activator, resveratrol. We found that treatment with CLBL and resveratrol stimulated lytic viral replication, suggesting that treatment of cells with agents that target cellular genes differentially expressed in latently infected cells can stimulate lytic replication. These findings may offer new insights into the interaction of the latently infected host cell and HIV and suggest therapeutic approaches for inhibiting HIV infection and for manipulating cells latently infected with HIV so as to trigger lytic replication.

scholarly journals A Theileria annulata DNA Binding Protein Localized to the Host Cell Nucleus Alters the Phenotype of a Bovine Macrophage Cell Line

2004 ◽  
Vol 3 (2) ◽  
pp. 495-505 ◽  
Author(s):  
Brian R. Shiels ◽  
Sue McKellar ◽  
Frank Katzer ◽  
Kim Lyons ◽  
Jane Kinnaird ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Cell ◽  
Cell Line ◽  
Mammalian Cells ◽  
Theileria Annulata ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Nuclear Localization Signals ◽  
Bovine Macrophage ◽  
Infected Cells

ABSTRACT The apicomplexan parasite Theileria annulata is the only intracellular eukaryote that is known to induce the proliferation of mammalian cells. However, as the parasite undergoes stage differentiation, host cell proliferation is inhibited, and the leukocyte is eventually destroyed. We have isolated a parasite gene (SuAT1) encoding an AT hook DNA binding polypeptide that has a predicted signal peptide, PEST motifs, nuclear localization signals, and domains which indicate interaction with regulatory components of the higher eukaryotic cell cycle. The polypeptide is localized to the nuclei of macroschizont-infected cells and was detected at significant levels in cells that were undergoing parasite stage differentiation. Transfection of an uninfected transformed bovine macrophage cell line, BoMac, demonstrated that SuAT1 can modulate cellular morphology and alter the expression pattern of a cytoskeletal polypeptide in a manner similar to that found during the infection of leukocytes by the parasite. Our findings indicate that Theileria parasite molecules that are transported to the leukocyte nucleus have the potential to modulate the phenotype of infected cells.

CTCF, a conserved nuclear factor required for optimal transcriptional activity of the chicken c-myc gene, is an 11-Zn-finger protein differentially expressed in multiple forms

1993 ◽  
Vol 13 (12) ◽  
pp. 7612-7624
Author(s):  
E M Klenova ◽  
R H Nicolas ◽  
H F Paterson ◽  
A F Carne ◽  
C M Heath ◽  
...  
Keyword(s):  
Dna Binding ◽  
Cell Lines ◽  
Gene Promoter ◽  
Differentially Expressed ◽  
Ctcf Binding ◽  
Ctcf Binding Site ◽  
Mobility Shift ◽  
Chicken Cell ◽  
Nuclear Extracts ◽  
Myc Gene

A novel sequence-specific DNA-binding protein, CTCF, which interacts with the chicken c-myc gene promoter, has been identified and partially characterized (V. V. Lobanenkov, R. H. Nicolas, V. V. Adler, H. Paterson, E. M. Klenova, A. V. Polotskaja, and G. H. Goodwin, Oncogene 5:1743-1753, 1990). In order to test directly whether binding of CTCF to one specific DNA region of the c-myc promoter is important for chicken c-myc transcription, we have determined which nucleotides within this GC-rich region are responsible for recognition of overlapping sites by CTCF and Sp1-like proteins. Using missing-contact analysis of all four nucleotides in both DNA strands and homogeneous CTCF protein purified by sequence-specific chromatography, we have identified three sets of nucleotides which contact either CTCF or two Sp1-like proteins binding within the same DNA region. Specific mutations of 3 of 15 purines required for CTCF binding were designed to eliminate binding of CTCF without altering the binding of other proteins. Electrophoretic mobility shift assay of nuclear extracts showed that the mutant DNA sequence did not bind CTCF but did bind two Sp1-like proteins. When introduced into a 3.3-kbp-long 5'-flanking noncoding c-myc sequence fused to a reporter CAT gene, the same mutation of the CTCF binding site resulted in 10- and 3-fold reductions, respectively, of transcription in two different (erythroid and myeloid) stably transfected chicken cell lines. Isolation and analysis of the CTCF cDNA encoding an 82-kDa form of CTCF protein shows that DNA-binding domain of CTCF is composed of 11 Zn fingers: 10 are of C2H2 class, and 1 is of C2HC class. CTCF was found to be abundant and conserved in cells of vertebrate species. We detected six major nuclear forms of CTCF protein differentially expressed in different chicken cell lines and tissues. We conclude that isoforms of 11-Zn-finger factor CTCF which are present in chicken hematopoietic HD3 and BM2 cells can act as a positive regulator of the chicken c-myc gene transcription. Possible functions of other CTCF forms are discussed.

Regulatory Proteins Bind at the Polymorphic NFSE Element of CYP3A4.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4453-4453
Author(s):  
Tal David-Kalish ◽  
Deborah Rund ◽  
Elad Malik ◽  
Sara Bar Cohen
Keyword(s):  
Dna Binding ◽  
Cell Lines ◽  
Blast Crisis ◽  
Binding Activity ◽  
Dna Probe ◽  
Luciferase Expression ◽  
Cytochrome P450 Enzyme ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Nuclear Extracts

Abstract CYP3A4 is the most abundant cytochrome P450 enzyme in the liver and is involved in the metabolism of most clinically used drugs. An A to G substitution in the nifedipine responsive element (NFSE) in the promoter of this gene has been found to be associated with a lower incidence of pediatric therapy-related leukemia (Felix, Proc Natl Acad Sci USA95:13176, 1998) and adult therapy-related leukemia (Rund et al, Leukemia, accepted for publication). To study the effect of this polymorphism on gene expression in hematopoietic cells, we constructed reporter plasmids with the luciferase gene (in pGL3E) under control of the CYP3A4 promoter, using both the polymorphic and normal sequences. These plasmids were transfected into several cell lines of hematopoietic origin and luciferase was quantitated. We used KG1a (myeloid leukemia), K562 (CML blast crisis), and as controls, MelA1, a melanoma line and HepG2, a hepatoma line. Experiments were repeated at least three times for each cell line. The results consistently demonstrated 20–30% lower luciferase activity (in KG1a and K562 respectively) using the polymorphic sequence as compared to the normal sequence while the MelA1 and HepG2 lines showed the opposite effect, a 25% higher luciferase expression with the variant sequence. The results for HepG2 were in agreement with those reported by Rebbeck (Environmental and Molecular Mutagenesis49:299, 2003). To identify the factors binding at NFSE which may influence expression, electrophoretic mobility shift assays were performed using nuclear extracts of both cell lines (K562, KG1a, and HL60) and patient leukemia cells with a DNA probe representing the normal and polymorphic sequences. A gel shift was demonstrated, indicating binding of nuclear extracts to the region of the polymorphism. The database of transacting factors states complete homology of the polymorphic sequence of the NFSE region with the consensus binding site of HSF-1. We therefore performed a series of experiments to determine if HSF-1 is the protein binding at that site. HSF-1 is a multimeric transcription factor which binds to heat shock elements in many promoters which are rapidly transcribed following stress by increases in temperature. We found that recombinant HSF-1 did not bind to the DNA probe alone. However, nuclear extracts of cells which underwent stress by heating to 43°C for one hour (which is known to increase HSF-1 production) demonstrated increased binding to the probe representing the region of the polymorphism and Western blotting demonstrated more HSF-1 in these extracts. Using a Streptavidin-biotin system with a DNA fragment representing the NFSE region, we demonstrated that DNA binding activity to the probe was present in the elution fractions which contained HSF-1, as detected by ECL (enhanced chemoluminescence). Elution fractions which did not show DNA binding activity did not contain detectable HSF-1. We conclude that HSF-1 may be the protein which binds at the NFSE element of the CYP3A promoter but that it binds either as a multimer or as part of a complex of several proteins, which complicates its detection as a DNA binding protein.

scholarly journals Adenoviral E2 IVa2 protein interacts with L4 33K protein and E2 DNA-binding protein

2013 ◽  
Vol 94 (6) ◽  
pp. 1325-1334 ◽  
Author(s):  
Yadvinder S. Ahi ◽  
Sai V. Vemula ◽  
Suresh K. Mittal
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Indirect Evidence ◽  
Dna Binding Protein ◽  
Immunogold Electron Microscopy ◽  
Genome Packaging ◽  
Nuclear Extracts ◽  
Infected Cells ◽  
Unique Vertex

Adenovirus (AdV) is thought to follow a sequential assembly pathway similar to that observed in dsDNA bacteriophages and herpesviruses. First, empty capsids are assembled, and then the genome is packaged through a ring-like structure, referred to as a portal, located at a unique vertex. In human AdV serotype 5 (HAdV5), the IVa2 protein initiates specific recognition of viral genome by associating with the viral packaging domain located between nucleotides 220 and 400 of the genome. IVa2 is located at a unique vertex on mature capsids and plays an essential role during genome packaging, most likely by acting as a DNA packaging ATPase. In this study, we demonstrated interactions among IVa2, 33K and DNA-binding protein (DBP) in virus-infected cells by in vivo cross-linking of HAdV5-infected cells followed by Western blot, and co-immunoprecipitation of IVa2, 33K and DBP from nuclear extracts of HAdV5-infected cells. Confocal microscopy demonstrated co-localization of IVa2, 33K and DBP in virus-infected cells and also in cells transfected with IVa2, 33K and DBP genes. Immunogold electron microscopy of purified HAdV5 showed the presence of IVa2, 33K or DBP at a single site on the virus particles. Our results provide indirect evidence that IVa2, 33K and DBP may form a complex at a unique vertex on viral capsids and cooperate in genome packaging.

scholarly journals Cellular localisation of the proteins of region 3 of feline enteric coronavirus

2018 ◽  
Vol 66 (3) ◽  
pp. 493-508 ◽  
Author(s):  
István Mészáros ◽  
Ferenc Olasz ◽  
Enikő Kádár-Hürkecz ◽  
Ádám Bálint ◽  
Ákos Hornyák ◽  
...  
Keyword(s):  
Dna Binding ◽  
Open Reading Frames ◽  
Target Cells ◽  
Transcription Activator ◽  
Cellular Localisation ◽  
Infected Cells ◽  
Natural Target ◽  
Golgi Network ◽  
Reading Frames

Feline enteric coronaviruses have three open reading frames (ORFs) in region 3 (3a, 3b, and 3c). All three ORFs were expressed with C-terminal eGFP and 3xFLAG tags in different cell lines and their localisation was determined. ORF 3a is predicted to contain DNA-binding and transcription activator domains, and it is localised in the nucleus and in the cytoplasm. ORF 3b is also predicted to contain DNA-binding and activator domains, and was found to localise in the mitochondrion. Besides that, in some of the non-infected and FIPV-infected cells nucleolar, perinuclear or nuclear membrane accumulation of the eGFP-tagged 3b was observed. The exact compartmental localisation of ORF 3c is yet to be determined. However, based on our co-localisation studies 3c does not seem to be localised in the ER-Golgi network, ERGIC or peroxisomes. The expression of 3c-eGFP is clearly cell type dependent, it is more stable in MARC 145 cells than in Fcwf-4 or CrFK cells, which might reflectin vivostability differences of 3c in natural target cells (enterocytes vs. monocytes/macrophages).

scholarly journals A Bovine Lymphosarcoma Cell Line Infected with Theileria annulata Exhibits an Irreversible Reconfiguration of Host Cell Gene Expression

PLoS ONE ◽  
2013 ◽  
Vol 8 (6) ◽  
pp. e66833 ◽  
Author(s):  
Jane H. Kinnaird ◽  
William Weir ◽  
Zeeshan Durrani ◽  
Sreerekha S. Pillai ◽  
Margaret Baird ◽  
...  
Keyword(s):  
Gene Expression ◽  
Host Cell ◽  
Cell Line ◽  
Theileria Annulata ◽  
Cell Gene Expression ◽  
Cell Gene

scholarly journals Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: inactivation of specific transcription factors.

1987 ◽  
Vol 7 (11) ◽  
pp. 3880-3887 ◽  
Author(s):  
L G Fradkin ◽  
S K Yoshinaga ◽  
A J Berk ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Rna Polymerase ◽  
Host Cell ◽  
Rna Polymerase Iii ◽  
Cell Protein ◽  
Polymerase Iii ◽  
Infected Cells

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoter, however, was not altered by infection of cells with the virus. We conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.

scholarly journals Semliki Forest Virus Nonstructural Protein 2 Is Involved in Suppression of the Type I Interferon Response

2007 ◽  
Vol 81 (16) ◽  
pp. 8677-8684 ◽  
Author(s):  
Lucy Breakwell ◽  
Pia Dosenovic ◽  
Gunilla B. Karlsson Hedestam ◽  
Mauro D'Amato ◽  
Peter Liljeström ◽  
...  
Keyword(s):  
Gene Expression ◽  
Host Cell ◽  
Semliki Forest Virus ◽  
Nonstructural Protein ◽  
Nuclear Localization Sequence ◽  
Type I ◽  
Viral Mutant ◽  
Cell Gene Expression ◽  
Infected Cells ◽  
Cell Gene

ABSTRACT The type I interferons (IFNs) are potent mediators of antiviral immunity, and many viruses have developed means to block their expression or their effects. Semliki Forest virus (SFV) infection induces rapid and profound silencing of host cell gene expression, a process believed to be important for the inhibition of the IFN response. In SFV-infected cells, a large proportion of the nonstructural protein nsp2 is found in the nucleus, but a role for this localization has not been described. In this work we demonstrate that a viral mutant, SFV4-RDR, in which the nuclear localization sequence of nsp2 has been rendered inactive, induces a significantly more robust IFN response in infected cells. This mutant virus replicates at a rate similar to that of the parental SFV4 strain and also shuts off host cell gene expression to similar levels, indicating that the general cellular shutoff is not responsible for the inhibition of IFN expression. Further, the rate of virus-induced nuclear translocation of early IFN transcription factors was not found to differ between the wild-type and mutant viruses, indicating that the effect of nsp2 is at a later stage. These results provide novel information about the mode of action of this viral IFN antagonist.

Sign in / Sign up

Export Citation Format

Share Document